In work supported by our original grant, we sought to identify and characterize surface antigens that are expressed by human mononuclear phagocytes as a consequence of activation. Toward this goal, we succeeded in generating a series of murine monoclonal antibodies specific for a surface determinant, Mo3 (p75,50), which is expressed by human monocytes and myelomoncytic cell lines after exposure to activating factors (LPS, MDP, certain cytokines, and phorbol compounds) in vitro. Surface Mo3 expression is also acquired by human umbilical vein endothelial cells after stimulation with phorbols in vitro, and by tissue macrophages endothelial cells within foci of inflammation in vivo. These data support the concept that Mo3 is a serological marker of activation, and, based on our studies to date, Mo3 represents the first well- characterized activation-associated antigen of the human mononuclear phagocyte system. In functional experiments, anti-Mo3 antibody blocks the response of monocytic cells to migration inhibitory factor (MIF) suggesting that Mo3 plays a rile in macrophage migration. The aims of this renewal application are to extend our knowledge about the cellular distribution, structure and function of Mo3. Specifically, we will: (1) Quantitate the Mo3 (125I-Fab) binding sites on human myelomonocytic cells before and after activation; (2) Determine the distribution of Mo3 expression among resting and inflammatory (activated) differentiated macrophages; (3) Extend our biochemical characterization of Mo3 to include an analysis of biosynthesis, amino acid and carbohydrate composition, and N-terminal amino acid sequence (as determined in part from an examination of antibody affinity-purified Mo3), and an evaluation of Mo3 phosphorylation, myristoylation, and structural heterogeneity. If current attempts at molecular cloning are successful, we will study the regulation of Mo3 expression at the RNA level. (4) Determine the subcellular localization of Mo3 by immunogold electron microscopy or analysis of Mo3 expression within subcellular fractions; (5) Determine if Mo3 is secreted or shed; (6) Examine the mechanism of Mo3 antigenic modulation; and (7) Asses the function of Mo3 by determining if (a) Mo3 is associated with the putative MIF receptor, (b) Mo3 plays a direct role in cellular adhesion and motility, and (c) Mo3 is a receptor by which signals are transduced.